Our goal is understanding the basis of RNA editing in trypanosomes. These organisms cause a number of debilitating and eventually lethal diseases in hundreds of millions of people, mainly in third world countries. Trypanosomes are very primitive eukaryotes and exhibit unique biochemical properties, potential therapeutic targets for these poorly treatable diseases. Probably the trypanosome's most novel feature, and surely the strangest form of RNA maturation known, is RNA editing. This editing creates mature mRNA coding regions by inserting U residues (often massive amounts) and less frequently deleting U residues in primary mitochondrial transcripts, directed by pairing with guide RNAs. We found that U deletional and U insertional editing co-purify: with each other, with all the implicated component activities -- gRNA-directed endonuclease, 3'-U exonuclease, terminal-U-transferase, and RNA ligase -- and with a complex of only seven proteins (approximately equimolar), two of which are RNA ligases. We have cloned the genes for all seven proteins and find by RNAi and genetic knock-out that five are essential. One ligase is needed to join in U deletion; the other ligase serves in U insertion but can be replaced by the former ligase, both in vivo and in vitro. The other component editing activities presumably arise from the identified proteins. Most of these proteins also appear structurally important in the complex. To better understand the catalytic and structural roles of these proteins, we propose a focused series of genetic approaches. Using mainly RNAi and over-expression, and assessing in vitro, should enable assigning which proteins are needed: for association of which other proteins, for catalyzing which activities, for the ordered passage of substrate RNA between the active sites, and for the unanticipated distinction of U deletion and U insertion substrates at the cleavage and the ligation steps. The second phase of the proposed studies extends these analyses to examine the structural and catalytic role of specific protein domains. By creating targeted mutations in conserved sequence domains and favoring editing complex formation, their roles can be assessed independently of other functions the protein may serve. We feel our advances during the previous granting periods form a strong foundation from which to further explore this unique form of RNA maturation.